Siderocalin (lipocalin 2), found in neutrophil granules, uterine secretions and secreted from epithelial cells in response to inflammation or tumorigenesis. is also an acute phase protein, with markedly elevated levels in the serum and the synovium during bacterial infection. Though implicated in diverse physiological processes, siderocalin's function was mysterious until our recent identification of specific, high-affinity ligands for this protein: bacterial phenolate-type ferric siderophores, such as enterochelin (aka enterobactin; KD = 0.4 nanomolar) and the mixed-type carboxymycobactin ferric siderophores. We therefore propose that siderocalin functions as an antibacterial agent, sequestering iron as ferric siderophore complexes, complementing the general anti-microbial iron-depletion strategy of the innate immune system. Supporting this hypothesis, we have found that siderocalin is a potent bacteriostatic agent in vitro under iron-limiting conditions and, when knocked-out, renders animals remarkably susceptible to bacterial infection. Siderocalin apparently uses a novel, degenerate recognition mechanism to cross-react with distinct types of siderophores, broadening the utility of the response. This functional hypothesis also rationalizes the association of some siderophores with virulence: by alternately utilizing siderophores with markedly reduced affinity for siderocalin, pathogens can escape siderocalin-mediated iron-deprivation. The goal of this project is to determine how siderophore-specific components of the innate immune response recognize microbial siderophores. In Aim 1, we propose continuing crystallographic, mutagenesis and biophysical studies of siderocalin, and a panel of natural, synthetic and designed siderophores, to fully delineate how this protein recognizes siderophores. Siderocalin is a member of the diverse lipocalin protein family. We have also identified four distantly-related murine and avian lipocalins that either demonstrably bind alternate spectrums of bacterial siderophores or are predicted to on the basis of modeling studies. In Aim 2, we propose analogous structural and biochemical studies of these proteins to similarly parse their recognition machinery. Ultimately, these studies will not only fully characterize this component of antibacterial immune surveillance, but will also allow continuing and future studies of virulence and pathogenesis - as well as defining the potential utility of these proteins as novel anti-bacterial therapeutics.